Somatic mutations in the PTEN (Phosphatase and Tensin homolog deleted on chromosome 10) gene are known to cause tumors in a variety of human tissues. In addition, germline mutations in PTEN are the cause of human diseases (Cowden disease and Bannayan-Zonana syndrome) associated with increased risk of breast and thyroid cancer (Nelen M R et al. (1997) Hum Mol Genet, 8:1383-1387; Liaw D et al. (1997) Nat Genet, 1:64-67; Marsh D J et al. (1998) Hum Mol Genet, 3:507-515). PTEN is thought to act as a tumor suppressor by regulating several signaling pathways through the second messenger phosphatidylinositol 3,4,5 triphosphate (PIP3). PTEN dephosphorylates the D3 position of PIP3 and downregulates signaling events dependent on PIP3 levels (Maehama T and Dixon J E (1998) J Biol Chem, 22, 13375-8). In particular, pro-survival pathways downstream of the insulin-like growth factor (IGF) pathway are regulated by PTEN activity. Stimulation of the IGF pathway, or loss of PTEN function, elevates PIP3 levels and activates pro-survival pathways associated with tumorigenesis (Stambolic V et al. (1998) Cell, 95:29-39). Consistent with this model, elevated levels of insulin-like growth factors I and II correlate with increased risk of cancer (Yu H et al (1999) J Natl Cancer Inst 91:151-156) and poor prognosis (Takanami I et al, 1996, J Surg Oncol 61(3):205-8). In addition, increased levels or activity of positive effectors of the IGF pathway, such as Akt and PI(3) kinase, have been implicated in several types of human cancer (Nicholson K M and Anderson N G (2002) Cellular Signalling, 14:381-395).
In Drosophila melanogaster, as in vertebrates, the Insulin Growth Factor Receptor (IGFR) pathway includes the positive effectors PI(3) kinase, Akt, and PDK and the inhibitor, PTEN. These proteins have been implicated in multiple processes, including the regulation of cell growth and size as well as cell division and survival (Oldham S and Hafen E. (2003) Trends Cell Biol. 13:79-85; Garafolo R S. (2002) Trends Endocr. Metab. 13:156-162; Backman S A et al. (2002) Curr. Op. Neurobio. 12:1-7; Tapon N et al. (2001) Curr Op. Cell Biol. 13:731-737). Activation of the pathway in Drosophila can result in increases in cell size, cell number and organ size (Oldham S et al. (2002) Dev. 129:4103-4109; Prober D A and Edgar B A. (2002) Genes & Dev. 16:2286-2299; Potter C J et al. (2001) Cell 105:357-368; Verdu J et al. (1999) Cell Biol. 1:500-506).
Collagen prolyl 4-hydroxylases catalyze the hydroxylation of proline in x-pro-gly triplets in collagens and in proteins with collagen-like sequences. Hydroxyproline residues are essential for the folding of collagen polypeptides into triple-helical molecules. Collagen prolyl 4-hydroxylases are tetramers of 2 alpha subunits, such as P4HA3, and 2 beta subunits. P4HA3 (procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alpha polypeptide III) is one of several different types of alpha subunits and provides the major part of the catalytic site of the active enzyme. Alternatively spliced transcript variants have been observed, but their full-length nature has not been determined. P4HA3 expression may play a role in atherosclerotis (Van Den Diepstraten, et al (2003) Cloning of a novel prolyl 4-hydroxylase subunit expressed in the fibrous cap of human atherosclerotic plaque. Circulation 108: 508-511).
The ability to manipulate the genomes of model organisms such as Drosophila provides a powerful means to analyze biochemical processes that, due to significant evolutionary conservation, have direct relevance to more complex vertebrate organisms. Due to a high level of gene and pathway conservation, the strong similarity of cellular processes, and the functional conservation of genes between these model organisms and mammals, identification of the involvement of novel genes in particular pathways and their functions in such model organisms can directly contribute to the understanding of the correlative pathways and methods of modulating them in mammals (see, for example, Mechler B M et al., 1985 EMBO J 4:1551-1557; Gateff E. 1982 Adv. Cancer Res. 37: 33-74; Watson K L., et al., 1994 J Cell Sci. 18: 19-33; Miklos G L, and Rubin G M. 1996 Cell 86:521-529; Wassarman D A, et al., 1995 Curr Opin Gen Dev 5: 44-50; and Booth D R. 1999 Cancer Metastasis Rev. 18: 261-284). For example, a genetic screen can be carried out in an invertebrate model organism having underexpression (e.g. knockout) or overexpression of a gene (referred to as a “genetic entry point”) that yields a visible phenotype. Additional genes are mutated in a random or targeted manner. When a gene mutation changes the original phenotype caused by the mutation in the genetic entry point, the gene is identified as a “modifier” involved in the same or overlapping pathway as the genetic entry point. When the genetic entry point is an ortholog of a human gene implicated in a disease pathway, such as IGFR, modifier genes can be identified that may be attractive candidate targets for novel therapeutics.
All references cited herein, including patents, patent applications, publications, and sequence information in referenced Genbank identifier numbers, are incorporated herein in their entireties.